Bacillus velezensis strain B26 modulates the inflorescence and root architecture of Brachypodium distachyon via hormone homeostasis

Plant growth-promoting rhizobacteria (PGPR) influence plant health. However, the genotypic variations in host organisms affect their response to PGPR. To understand the genotypic effect, we screened four diverse B. distachyon genotypes at varying growth stages for their ability to be colonized by B. velezensis strain B26. We reasoned that B26 may have an impact on the phenological growth stages of B. distachyon genotypes. Phenotypic data suggested the role of B26 in increasing the number of awns and root weight in wild type genotypes and overexpressing transgenic lines. Thus, we characterized the expression patterns of flowering pathway genes in inoculated plants and found that strain B26 modulates the transcript abundance of flowering genes. An increased root volume of inoculated plants was estimated by CT-scanning which suggests the role of B26 in altering the root architecture. B26 also modulated plant hormone homeostasis. A differential response was observed in the transcript abundance of auxin and gibberellins biosynthesis genes in inoculated roots. Our results reveal that B. distachyon plant genotype is an essential determinant of whether a PGPR provides benefit or harm to the host and shed new insight into the involvement of B. velezensis in the expression of flowering genes.


Results
Bacterial inoculation elicited varied growth response of B. distachyon accessions. A differential response was observed in Bd21, Bd21-3, Bd18-1 and Bd30-1 in response to B26 colonization (Fig. 1a). At 14 days post inoculation (dpi), a significant increase of 150% in the number of awns and 250% increase in the shoot weight of inoculated accession Bd21 compared to non-inoculated control was observed (Fig. 1b, Table S2). The plant height and number of leaves of inoculated Bd18-1 increased by 34% and 78%, respectively compared to the control. At 28 dpi, Bd21-3 showed a significant increase in all growth parameters compared to the control (Fig. 1c). While Bd 30-1 at 28dpi, did not show a significant response to B26 inoculation as indicated by the growth parameters (Fig. 1c). However, there was no difference in flowering time of inoculated and noninoculated plants. Control and inoculated accessions flowered at the same time but an increase in the number of awns was observed.
B. distachyon accessions sustained populations of strain B26 in root and shoot tissues. Quantification was done in roots and shoots of B. distachyon accession Bd21-3 that responded well to B26 inoculation in terms of growth parameters, and accession Bd30-1 that showed similar growth responses to B26 as the control after 14 dpi and 28 dpi (Table 1). Strain B26 had similar sustaining endophytic populations in roots and shoots in both genotypes. In the case of Bd21-3, more copies were found in roots at 28dpi as compared to shoots (Table 1). On the contrary, Bd30-1 had more copies in shoots at 14dpi. However, more B26 gene copies were found in tissues of Bd21-3 as compared to Bd30-1.

Strain B26 improves root and shoot weights of transgenic lines. Detection of transgene in
UBI:FT1 and UBI:VRN1 was done by PCR. cDNA specific forward primer and pANIC vector AcV5 tag reverse primer were used to detect transgene in transgenic lines. PCR with VRN1-F /FT1-F and AcV5 tag yielded an expected band size of approximately 260 bp and 500 bp, respectively which confirmed the presence of transgene (Fig. S1a,b). No amplification was observed in wild type Bd21-3 as there is no transgene present. A wide differential growth response among the transgenic lines compared to the wild type genotype Bd21-3 was observed (Fig. 3a). At 28 dpi, the root and shoot weights of transgenic line UBI:FT1 significantly increased by 132% and 162%, respectively in response B26 (Table S3). Growth parameters such as the number of awns, root and awn weight of the wild type genotype Bd21-3 increased significantly by 34%, 52% and 43%, respectively (Fig. 3b Transcript abundance of flowering genes in roots and leaves of inoculated transgenic lines. At 28 dpi, the phenotypic observations of flowering transgenic lines (Fig. 3a, b) showed the effect of inoculation is more noticeable in roots and awns of transgenics. This prompted us to study the expression of flowering genes in both roots and shoots of transgenic lines at this growth stage. Each transgenic line was compared with the wild type separately. A significant upregulation in transcripts of FT1 gene (17,981-fold) was observed in inoculated roots of UBI:FT1 relative to non-inoculated wild type. Strain B26 did not induce FT1 nor VRN1 genes in shoot tissues of inoculated transgenic plants. However, transcripts levels of VRN1 gene were down-regulated in both roots and shoots of UBI:FT1 and UBI:VRN1 compared to the inoculated wild type (Fig. 4).  www.nature.com/scientificreports/    were the most abundantly detected phytohormones. The phytohormone homeostasis in Bd21-3 was significantly affected by B26. Growth promotion of the wild type Bd21-3 by strain B26 is significantly (p < 0.05) associated with increases in GA 4 (2-fold). While the amount of GA 7 , and IAA were significantly less by 4.8 and 2.3-fold ,respectively as compared to control roots (Fig. 5). In case of UBI:FT1, GA 1 was significantly higher in inoculated roots than control. However, the concentration of other phytohormones was detected less in inoculated UBI:FT1 roots as compared to control roots. In contrast, levels of GA 1 , GA 7 and IAA were 2.75, 1.59 and 1.89 times respectively higher significantly in inoculated roots of UBI:VRN1 when compared to control roots. However, Kinetin, Zeatin and GA 3 were below the detection level.
Transcript abundance of genes related to phytohormones in Bd21-3. A significant upregulation in transcripts of genes related to auxin biosynthesis was observed in wild type Bd21-3 only. Anthranilate synthase alpha subunit 1(ASA1) which catalyses the rate-limiting step of tryptophan biosynthesis 27 and Indole-3-acetic acid inducing gene (IAA18) were significantly up-regulated by 2.3 and 4.9-fold, respectively in inoculated roots as compared to control roots (Fig. 6). A significant downregulation was observed in transcript abundance of GA20ox1 which encodes gibberellin 20-oxidase enzyme that is involved in the later steps of the gibberellins (GA) biosynthesis pathway 28 Interestingly, DELLA proteins, a key negative regulator of GA signalling 29 was significantly up-regulated by 3.8-fold in inoculated roots as compared to control roots (Fig. 6).

Discussion
The data presented here indicate that B. distachyon is a useful model to study PGPR-plant association and could serve as a model for rice and wheat. A central finding in this study is that plant genotype is a crucial determinant of whether rhizobacteria inoculation promotes plant growth or not. The four genotypes behaved differently throughout the whole life cycle of the plants for each growth parameter and showed statistically positive or negative responses for one or more of the parameters tested. Such response is exemplified in genotype Bd21 and Bd21-3 in which induction of flowering was accelerated in response to B26. These results are not uncommon among plant accessions since naturally occurring resistance is common in studies of plant-microbe interactions. B. distachyon genotypes demonstrated significant and varied responses to infection by pathogenic insects and fungi 30 . Moreover, several B. distachyon genotypes differed in their ability to associate with two diazotrophic strains and several genotypes responded negatively to the strains 9 . Also, wild accessions of Arabidopsis thaliana showed reduced growth in response to Pseudomonas fluorescence 8 . Of interest, genotype Bd30-1 which performed less favourably among the other 3 accessions, had sustained B26 populations in roots and shoots, but  www.nature.com/scientificreports/ was insufficient to induce growth promotion in accession Bd30-1. This suggests that a different mechanism is implicated, and this requires further analysis. Molecular studies on the regulation of flowering genes (FT1, FT2, VRN1 and VRN2) in response to environmental cues have been intensively studied in Arabidopsis, cereals 31,32 , and B. distachyon 17 . However, molecular studies on the regulation of flowering genes in response to rhizobacteria are scarce 26,33 . Flowering in B. distachyon is mostly regulated by three key genes viz., VERNALIZATION1 (VRN1), VRN2, and FLOWERING LOCUS T (FT). VRN1, VRN2 and FT form a regulatory loop in wheat and barley [34][35][36] . We focused on studying transcript levels of flowering genes in Bd21-3 a genotype known as rapid flowering and Bd30-1 a genotype known to show intermediate flowering. The inoculation of genotype Bd21-3 with strain B26, induced an abundance of FT1 transcript levels in shoots and it was not a limiting factor in the upregulation of VRN1. Our results are in agreement with the elevated expression patterns of FT1 and VRN1 in the rapid flowering B. distachyon accessions 17 . Intriguingly, this trend supports the proposed model for wheat and barley during cold exposure 37,38 . However, VRN2 acts as a repressor of flowering and was expressed at lower levels in spring accession of wheat and barley 19 . In B. distachyon, VRN2 was also expressed at lower levels in the spring accession Bd21-3 20 . The current study supports this evidence since VRN2 was down-regulated in Bd21-3 accession line. In the case of the intermediate flowering accession line, Bd30-1, the expression of VRN2 was remarkably high compared to Bd21-3. Similar results were obtained by Ream et al. 17 in which Bd2-3 had more amounts of BdVRN2 and less amount of BdFT1, suggesting that VRN2 may play a role as a flowering repressor. Both Bd2-3 and Bd30-1 belongs to the Intermediate rapid flowering class.
To fully understand the role of B26 inoculation on flowering genes, we tested overexpressing flowering transgenic lines UBI:FT1 and UBI:VRN1. Phenotypic data suggested an increase in awn and root weights in inoculated transgenic plants. This triggered us to investigate flowering genes in roots in response to B26. Numerous flowering genes are identified in roots but were solely studied in the shoots. Bouché et al. 22 reported that flowering genes in the roots of Arabidopsis are differentially expressed during flowering and concluded that roots may be involved in flowering by sending systemic signals or may participate actively in the regulation of flowering genes. However, the causal relationship was not very well established. In our study, the increase in expression of FT1 in inoculated roots of UBI:FT1 positively correlates with root weight. These transgenes are expressed under the control of maize ubiquitin constitutive promoter 17 which upregulates the flowering gene expression, irrespective of bacterial treatment. Hence the increase in the transcript of FT1 in inoculated roots of UBI:FT1 is solely due to B26 inoculation. These results indicate that strain B26 modulates the transcription of flowering genes. This is the first report, according to our knowledge, that rhizobacteria can induce flowering genes in B. distachyon roots.
Non-symbiotic rhizobacteria contribute beneficial traits to colonized plants through bioactive compounds including, phytohormones 39 . These phytohormones influence the physiological processes of plants at very low levels 40 . Indeed, many studies demonstrated that rhizobacteria is associated with phytohormone concentrations and involved in homeostasis such as IAA, gibberellins, and IBA 41 . In our study, the endogenous phytohormones concentrations in the roots were modified by strain B26. Surprisingly, the concentrations of IAA and GA 7 in inoculated Bd21-3 were lower than the control, but the transcripts of IAA were moderately up-regulated. This might be interpreted that strain B26 positively affected plant growth via metabolizing these phytohormones in the soil, a widespread trait among soil bacteria 42 . This plant hormonal homeostasis may rise from microbial consumption and production of hormones or fluctuations in plant hormones in planta 41 . Thus, plant-associated microbes can modulate plant metabolism by altering the plant hormone levels. Indeed, improved root growth of inoculated transgenic line UBI:FT1 is attributed to GA 1 production and in UBI:VRN1 to GA 7 and IAA. There is considerable evidence that gibberellins in grasses influence flower initiation 43 Given that B26 affected endogenous amounts of phytohormones, the question then arises whether B26 effects on wild and transgenic lines resulted in larger root volume. We examined the roots of wild type and transgenic lines by Macro CT scanning that were inoculated with B26 and compared them to the control. Consistent with the induction of phytohormones in inoculated wild and transgenic lines, B26 had a positive effect on root volume of all accession lines. These results are congruent with preceding data and provide additional evidence of phytohormone modulation in Brachypodium roots by B26.
In summary, this report offers novel information about the long-term effects of a PGPR on plant development, advancing the knowledge on these relevant biological interactions. Our study shed new light on the involvement of strain B26 by influencing the flowering process in the roots. Key causal relationships cannot be established since we know little about the expression role of flowering genes in the Brachypodium roots and how they are connected to above-ground tissues. We also conclude that plant genotypes are critical to a successful interaction with PGPR.

Bacterial strain, growth, and inoculum preparation. The Plant Growth Promoting Rhizobacteria
(PGPR) viz., Bacillus velenzensis strain B26 44 , formally known as B. subtilis 4 was used in this study. The strain B26 was stored in 20% glycerol stocks in Lysogeny Broth (LB) (BDH chemical Ltd, Mississauga, ON, Canada) at − 80 °C. Revival of strain B26 was done on LB at 28 ± 1.0 °C on a rotatory shaker at 120 rpm until an OD 600 of 1.0 (10 6 CFU mL −1 ) was reached. Cells of strain B26 were centrifuged, washed, and suspended in a volume of phosphate buffer (1 M, pH 7) and used as inoculum for all experiments.

Plant material and growth conditions of wild type and transgenic lines. Four Brachypodium dis-
tachyon accessions were selected based on their origins, vernalization requirements and flowering time. Selected accessions were Bd21, Bd21-3, Bd18-1 and Bd30-1 (Table S1). Wild type seeds were provided by Dr Jean-Benoit Growth conditions of wild-type B.distachyon accessions: Seeds were sterilized following the methodology of Vain et al. 47 . Stratification and vernalization of seeds were done by placing them between two moist filter papers in a Petri dish and incubating them at 4 °C in the dark. The number of days for seed incubation was decided according to the vernalization requirement of wild type accessions (Table S1)

Genotyping of Transgenic lines.
To confirm the homozygosity of transgenic lines, PCR-based genotyping was carried out. DNA was extracted from young leaves of transgenic plants following the modified CTAB method. cDNA specific forward primer and pANIC vector AcV5 tag reverse primer were used to detect transgene (Table 3). Wild type Bd21-3 was used as control. The presence and absence of amplification confirmed the transgene. Single-band amplification was considered a homozygous plant containing transgene. Only homozygous plants were used.

B26 Inoculation and Assessment of Plant Growth Parameters of Wild type Accessions and
Transgenic Lines. Experiment 1. To examine the differential response of B. distachyon to B26 inoculation, wild accession lines were inoculated with strain B26 at defined phenological growth stages using BBCH numerical scale 49 . Twenty-one days old plants (BBCH 23) were inoculated with 10 mL of B26 cells suspended in phosphate buffer (10 6 CFU mL −1 ), while control plants received 10 mL of phosphate buffer per pot. Plants were harvested after 14 and 28 days post-inoculation (dpi) at defined phenological (BBCH 61) and (BBCH73) growth stages, respectively, and various phenotypic parameters were recorded. Five pots were harvested at each harvesting time point by carefully removing the substrate and washing the roots carefully. Growth parameters including Plant height, number of leaves, awns, tillers, fresh root and shoot weight were recorded. At each harvesting stage leaf and root samples were collected and stored at − 80 °C for downstream applications. The experiment was repeated twice.

Experiment 2.
To determine the effect of inoculation on B. distachyon flowering, overexpressing transgenic lines were observed for plant growth parameters. 14-days old (BBCH 13) transgenic lines and wild type Bd21-3 were inoculated with 10 mL of B26 inoculum as described in the previous section. Data was recorded after 14 dpi (BBCH53), 28 dpi (BBCH69) and 42 dpi (BBCH87). At each harvesting time point, data of 5 pots per accession were recorded for plant height, number of leaves, awns, tillers, awn weight, fresh root and shoot weight. At each harvesting stage leaf and root samples were collected and stored at − 80 °C for downstream applications.

Experiment 3.
To compare the total root volume between control and inoculated plants, macro CT-Scanning was done. A Semi-hydroponics system was developed for scanning of roots using Magenta GA-7 tissue culture boxes that were filled with sterile glass low alkali beads (Ceroglass, USA) saturated with Hoagland's solution as fully described in Sharma et al. 5 . Pre-germinated seeds of wild type Bd21-3, transgenic lines UBI:FT1 and UBI:VRN1 (6 seeds/box) were transferred to Magenta boxes where each box is an experimental unit. Boxes were incubated in a controlled growth cabinet (Conviron, Canada) with light intensity of 300 μmoles m 2 /s,16 h light and 8 h dark at day/night temperatures of 21 °C/18 °C. After 14 days of growth, three boxes of each line received B26 inoculum (500 µL OD 600 of 1) suspended in phosphate buffer (1 M, pH), and three control boxes received 500 μL of phosphate buffer alone. All boxes were incubated in a controlled growth cabinet. A total of 6 Magenta boxes were used per line.

B26 quantification in root and leaves of selected wild type B. distachyon accessions.
Quantification of B26 DNA copy number was performed in roots and leaves of Bd21-3 and Bd30-1 at 14, and 28 dpi using qPCR. Genomic DNA was extracted from 1 g of powdered tissue using the modified CTAB method. DNA from the pure culture of B26 was also extracted from a single B26 colony using the boiling method 50 . For detection purposes, conventional PCR was done using B26 strain-specific primers in inoculated leaves and roots of selected accessions. B26 bacterial DNA served as a positive control in PCR. Cloning and qPCR reactions were www.nature.com/scientificreports/ performed as described in Gagne-Bourque et al. 3 . To calculate the quantity of bacterial DNA in inoculated roots and leaves, Cq (Cycle quantification) values of plant DNA were correlated with Cq values in the standard curve. Moreover, for reliability of the designed method, correlation coefficient and the amplification efficiency were calculated from the formula X o = E AMP (b-Cq) = 10 (Cq-b)/m)) , where X o = initial reaction copies, E AMP = Exponential amplification, b = y-intercept of the standard curve (log 10 of copies), m = slope of standard curve.

Phytohormone analysis.
To determine the effect of inoculation on phytohormones, endogenous levels of plant phytohormones including auxin, cytokinin, gibberellins and abscisic acid was measured using the modified protocol of Li et al. 51 . Inoculated and control roots of Bd21-3, transgenic lines; UBI:FT1 and UBI:VRNI1 from Experiment 2 were subjected to phytohormone analysis after 28dpi. Root samples were crushed in liquid nitrogen. Samples were sent in triplicates to The Metabolomics Innovation Centre, UVic-Genome BC Proteomics Centre, Victoria, BC, Canada. Briefly, 100 mg of each sample was precisely weighed into a 2-mL safe-lock Eppendorf tube. 4 µL of 5% formic acid in water per mg of raw tissue and two 4-mm stainless steel balls were added. The sample was homogenized at a shaking frequency of 30 Hz on a MM 4000 mixer mill for 1 min three times. Methanol, at 16 µL per mg raw tissue was then added. The sample was homogenized again for 1 min three times, followed by sonication in an ice-water bath for 5 min and centrifugal clarification at 21,000g and 10 °C for 10 min. The clear supernatant was collected for the analysis of auxins, cytokinin, gibberellins and abscisic acid. Phytohormones were analysed with UPLC-multiple-reaction monitoring (MRM) mass spectrometry on an Agilent 1290 UHPLC coupled to an Agilent 6495B QQQ mass spectrometer equipped with an ESI source which was operated in the negative-ion mode. LC separation was carried out on a C18 UPLC column (2.1 × 150 mm, 1.8 µm). Concentrations of the detected compounds in the sample solutions were calculated by interpolating the constructed linear-regression calibration curve with the measured analyte-to-internal standard peak area ratios.
CT scanning of wild type Bd21-3 and transgenic lines. The total root volume of inoculated and noninoculated wild accession Bd21-3, transgenic lines UBI:FT1 and UBI:VRN1 grown in magenta boxes were compared by performing macro CT-scanning at 28 dpi. The root systems were scanned using macro-CT scanning with the Canon CT Aquilion Prime SP at the CT Scanning Laboratory for Agricultural and Environmental Table 3. List of primers used in this study. RNA extraction, cDNA synthesis and qRT-PCR analysis. Transcript abundance of flowering genes in selected B. distachyon wild type and transgenic lines. In response to B26 inoculation, we decided to choose the best phenotypic performer in terms of growth parameters (Bd21-3) and the least phenotypic performer (Bd30-1). We examined the gene expression of Brachypodium flowering pathway genes viz., FT1, FT2, VRN1 and VRN2 in leaves of Bd21-3 and Bd30-1 from Experiment 1 at 14 dpi and 28 dpi. To study the genotypic response of B26 on B. distachyon transgenic lines, transcript abundance of FT1 and VRN1 was measured in control and inoculated transgenic lines; UBI:FT1 and UBI:VRN1 along with wild type Bd21-3 roots and leaves from Experiment 2 at 28dpi. Briefly, total RNA was extracted from flash-frozen pulverized 100 mg of inoculated and control tissues using Spectrum™ Plant Total RNA Kit (Sigma Aldrich, US) following the manufacturer's protocols. One Script RT ABM kit (Vancouver, Canada) was used for reverse-transcription of RNA (500 ng) following the manufacturer's protocols. PCR assays were performed on three biological replicates and two technical replicates. Primer details are present in Table 3. The conditions for qRT-PCR were adjusted for each primer set. PCR amplification was performed in a 10 µL reaction following the protocol of Sharma et al. 5 . The 2 −ΔΔCT method 52 was applied to normalize the target gene over the housekeeping genes UBC18. Bestkeeper tool was used to compare housekeeping genes UBC18 and ACTIN2. UBC18 had the lowest coefficient variation as compared to ACTIN2 so UBC18 was chosen for the normalization.

Gene of interest
Transcript abundance of genes encoding phytohormones in Bd21-3. The effect of B26 inoculation on the phytohormone production by B.distachyon roots was quantified using qRT-PCR. Transcript abundance of auxin and gibberellins biosynthesis genes was measured only in roots of Bd21-3 from Experiment 2 at 28 dpi. Primer sets (Table 3) were designed based on gene sequences retrieved from Phytozome Bd21-3 v1.1 genome (Phytozome v12.1, https:// phyto zome. jgi. doe. gov/ pz/ portal. htmL. Primers were designed online from IDT website using Primer Quest Tool (https:// www. idtdna. com/ Prime rQuest/ Home/ Index). To confirm the specificity of Primers, sequences were checked for hairpins and hetero-dimer formations using the Oligoanalyzer tool (http:// www. idtdna. com/ calc/ analy zer) and submitted to Nucleotide Blast at NCBI (http:// www. ncbi. nlm. nih. gov/) and were custom synthesized by Integrated DNA Technologies (IDT, Iowa, USA). One hundred milligrams of tissue was subjected to RNA extraction. cDNA preparation and qRT-PCR were performed as described in the previous section.
Statistical analysis. Data of all experiments were analysed using IBM Statistics SPSS Version 24(SPSS Inc., Chicago, IL). Comparison of means was performed by independent student t-test for comparison between control and inoculated samples. Tukey's test was performed to compare the means of multiple treatments. We considered a p < 0.05 acceptable for statistical significance. Experiments 1 and 2 were performed using 5 replicates for each control and inoculated pots. To prevent contamination of treatments, two growth chambers were used for control and inoculated plants. To study the confounding effect of growth chambers, the experiments were repeated twice by exchanging the growth chambers of treatment with control plants.